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Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo. In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo. Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer. IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo. In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo. Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28(+)/CD95(+) central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer. IMPORTANCE Adoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

The late (L) domain sequence used by mouse mammary tumor virus (MMTV) remains undefined. Similar to other L domain-containing proteins, MMTV p8 and p14 NC proteins are monoubiquitinated, suggesting L domain function. Site-directed mutagenesis of p8, PLPPV, and p14 NC , PLPPL, sequences in MMTV Gag revealed a requirement only for the PLPPV sequence in virion release in a position-dependent manner. Electron microscopy of a defective Gag mutant confirmed an L domain budding defect morphology. The equine infectious anemia virus (EIAV) YPDL core L domain sequence and PLPPV provided L domain function in reciprocal MMTV and EIAV Gag exchange mutants, respectively. Alanine scanning of the PLPPV sequence revealed a strict requirement for the valine residue but only minor requirements for any one of the other residues. Thus, PLPPV provides MMTV L domain function, representing a fourth type of retroviral L domain that enables MMTV Gag proteins to co-opt cellular budding pathways for release.

Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses. Fifty six of the 82 ORFs were recognized by at least one individual but there was little overlap between participants. Responses to at least one ORF pool were observed in all 19 patients and in 7 seropositive donors. Four seropositive donors and 10 seronegative donors had no detectable responses while 3 seronegative donors had weak responses to one ORF. Patients recognised more ORFs than the donors (p=0.04) but the response intensity (spot forming units: SFU per million cells) was similar in the two groups. In four of the responding donors, individual peptides eliciting the predominant responses were identified: three donors responded to only one peptide per ORF, while one recognized five. Using intracellular cytokine staining in four participant samples, we detected peptide-induced IFN-γ, MIP1-β, and TNF-α as well as CD107a degranulation, consistent with multifunctional effector responses in CD8+ and CD4+ T cells. Sequence analysis of TCRs present in peptide specific T-cell clones generated from two participants showed both mono- and multi-clonotypic responses. Finally, we molecularly cloned the KSHV specific TCRs and incorporated the sequences into retroviral vectors to transfer the specificities to fresh donor cells for additional studies. This study suggests that KSHV infected individuals respond to diverse KSHV antigens, consistent with a lack of shared immunodominance and establishes useful tools to facilitate KSHV immunology studies.

Follicular helper CD4 T cells, T FH , residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8 hCXCR5 ) exhibited ligand-specific signaling and chemotaxis in vitro . Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8 hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8 hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8 hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8 hCXCR5 T cells were present throughout the follicles with some observed near infected T FH . In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication. IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, T FH , present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles, and those that do fail to robustly control viral replication in the T FH population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on T FH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus.

Human immunodeficiency virus type 1 (HIV-1) Gag-RNA interactions are required for virus assembly. However, our prior study found that a defect in particle production exhibited by an HIV-1 proviral mutant with a severe deletion in the RNA-binding nucleocapsid (NC) region of Gag, NX, could be reversed by eliminating its protease activity. While our follow-up study indicated that a secondary RNA-binding site in Gag can also provide the required RNA-binding function, how protease activity inhibits NX virion production is still unclear. Therefore, we tested three possible mechanisms: NX virions are unstable and fall apart after budding; NX Gag assembly is slowed, allowing protease processing to start before particle formation; or the protease region within NX Gag-Pol becomes activated prematurely and processes the assembling Gag. We found that NX particles were as stable as wild-type virions. Furthermore, even a modest slowing of protease activity could rescue NX. Pulse-chase analysis revealed that the initial particle production by NC-deleted Gag was delayed compared to that of wild type Gag, but once started, the rate of production was similar, revealing a defect in the initiation of assembly. Wild-type Gag particle production was not eliminated or decreased in the presence of excess NX Gag-Pol, inconsistent with a premature activation of protease. Overall, these results indicate that the particle formation defect of NX is due to delayed initiation of assembly caused by the absence of NC in Gag, making it vulnerable to protease processing before budding can occur. Therefore, NC plays an important initiating role in Gag assembly.

CD8 + T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8 + T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4 + T cells. We used a set of SIV mac 239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I high or MHC class I intermediate . However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I low ) despite the observations that all CTL clones showed similar IFN-γ responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.

To study CD4 + T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4 + T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8 + T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1β, and CD107a degranulation. Gag68 effectively suppressed the spread of SIV mac239 in CD4 + T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4 + effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4 + T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4 + T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4 + T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model.

The HIV-1 nucleocapsid protein (NC) has been hypothesized to be cleaved by the viral protease (PR) during early infection. Characterization of viruses, with amino-acid substitutions that modulate PR cleavage of NC in vitro, was performed in cell culture. Two of the NC mutants, NCN17F and NCN17G, had decreased infectivity and exhibited severe H9 replication defects. Examination of viral DNA after infections revealed defects in reverse transcription and integration, although integration defects were cell-type dependent. However, while the defects in reverse transcription and integration correlate with lowered infectivity in a single-round of infection, they did not approach the magnitude of the replication defect measured in H9 cells over multiple rounds. Importantly, we fail to see evidence that H9 cells are re-infected with the NCN17G and NCN17F viruses 24 h after the initial infection, which suggests that the principal defect caused by these NC mutations occurs during late events of viral replication.

RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.

Retroviral late (L) domains present within Gag act in conjunction with cellular proteins to efficiently release virions from the surface of the cell. Three different critical core sequences have been identified as required elements for L-domain function: PPPY, PTAP (also PSAP), and YPDL, with different retroviruses utilizing one or two of these core sequences. The human immunodeficiency virus type 1 (HIV-1) L domain is centered around a PTAP sequence in the p6 region of Gag. To assess the ability of heterologous L-domain sequences to be functionally interchanged for those in full-length HIV-1, we produced a series of constructs that replaced PTAP-containing p6 Gag sequences with those of PPPY- or YPDL-based L domains. While previous studies had found that L domains are interchangeable in other retroviruses, most of the sequences introduced into p6 Gag failed to substitute for PTAP-mediated L-domain function. One exception was the 11-amino-acid p2b sequence of Rous sarcoma virus (RSV) Gag, which could fully restore HIV-1 budding, while a PPPPY sequence exchange alone did not. This suggests that the RSV L domain consists of more than simply its core L-domain sequence. The HIV-p2b chimera was as infectious as the wild type, produced normal virions, and was sensitive to proteasome inhibitors. These results show that L-domain sequences are not necessarily interchangeable. Thus, HIV-1 Gag might have a more stringent requirement for L-domain function than the other retroviruses previously studied.

Among the many host cell-derived proteins found in human immunodeficiency virus type 1 (HIV-1), HLA class II (HLA-II) appears to be selectively incorporated onto virions and may contribute to mechanisms of indirect imunopathogenesis in HIV infection and AIDS. However, the amount of HLA-II on the surface of HIV-1 particles has not been reliably determined due to contamination of virus preparations by microvesicles containing host cell proteins, including HLA-II. Even rigorous sucrose density centrifugation is unable to completely separate HIV-1 from microvesicles. CD45, a leukocyte integral membrane protein, is found on microvesicles, yet appears to be excluded from HIV-1 particles. Exploiting this observation, we have developed a CD45-based immunoaffinity depletion method for removing CD45-containing microvesicles that yields highly purified preparations of virions. Examination of CD45-depleted HIV-1 MN by high-pressure liquid chromatography, protein sequencing, and amino acid analyses determined a molar ratio of HLA-II to Gag of 0.04 to 0.05 in the purified virions, corresponding to an estimated average of 50 to 63 native HLA-II complexes (i.e., a dimer of α and β heterodimers) per virion. These values are approximately 5- to 10-fold lower than those previously determined for other virion preparations that contained microvesicles. Our observations demonstrate the utility of CD45 immunoaffinity-based approaches for producing highly purified retrovirus preparations for applications that would benefit from the use of virus that is essentially free of microvesicles.

Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9 Gag proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors. Lentiviruses and type C retroviruses assemble in association with the host plasma membrane, forming a bud that is released from the cell to produce a virion (46). The late assembly domain (L) within Gag is crucial for the efficient release of the budding virus from the plasma membrane (42). Three different sequences have been shown to possess L domain function: PPPY, found in Rous sarcoma virus (RSV) (51, 52), murine leukemia virus (MuLV), (54), and Mason-Pfizer monkey virus (53); PTAP, found in human immunodeficiency virus type 1 (HIV-1) (presumably P[T/S]AP for HIV-2 and simian immunodeficiency virus [SIV]) (11, 18); and YPDL, found in equine infectious anemia virus (EIAV) (34). Deletion or replacement of these sequences causes virions to mostly remain attached to the plasma membrane by a thin tether and to fail to separate from the cell. These L domain sequences can interact directly with cellular proteins (8, 9, 12, 13, 19, 35, 45), suggesting potential cellular partners for virus budding. Despite these findings, the pathway(s) used by retroviruses for budding is mostly unknown, though recent results suggest that components of the vacuolar protein sorting pathway might be used by HIV-1 (9). Experiments with several retroviruses have shown that Gag interacts with the ubiquitination pathway and that efficient budding requires active proteasomes (47). Here we examine EIAV for interactions with the ubiquitin (Ub)-proteasome system. EIAV particles contain free Ub and Ub-Gag conjugates. For

Our previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6 Gag and MuLV p12 Gag , that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1 NL4-3 p6 Gag and both lysines in Moloney MuLV p12 Gag . HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6 Gag , K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6 Gag or MuLV p12 Gag is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6 Gag does not affect the short-term release of virus from the cell, the maturation of Pr55 Gag , or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55 Gag can be monoubiquitinated, suggesting that p6 Gag is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6 Gag , showing that the ubiquitin inside the virus is not initially brought in as a p6 Gag conjugate. Although our results establish that monoubiquitination of p6 Gag and p12 Gag is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding.

Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1α, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X 2 -Cys-X 4 -His-X 4 -Cys-) designated retroviral (CCHC) Zn 2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn 2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn 2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn 2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn 2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed ∼5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/β-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn 2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn 2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn 2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.

All retroviruses (except the spumaretroviruses) contain a nucleocapsid (NC) protein that encodes one or two copies of the Zn2+-finger sequence -Cys-X2-Cys-X4-His-X4-Cys-. This region has been shown to be essential for recognition and packaging of the genomic RNA during virion particle assembly. Additionally, this region has been shown to be involved in early infection events in a wide spectrum of retroviruses, including mammalian type C [e.g., murine leukemia virus (MuLV)], human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus, and other retroviruses. Mutations in the two Zn2+-fingers of the NC protein of simian immunodeficiency virus strain Mne [SIV(Mne)] have been generated. The resulting virions contained the normal complement of processed viral proteins with densities indistinguishable from wild-type SIV(Mne). All of the mutants had electron micrograph morphologies similar to those of immature particles observed in wild-type preparations. RNA packaging was less affected by mutations in the NC protein of SIV(Mne) than has been observed for similar mutants in the MuLV and HIV-1 systems. Nevertheless, in vitro replication of SIV(Mne) NC mutants was impaired to levels comparable to those observed for MuLV and HIV-1 NC mutants; replication defective NC mutants are typically 10(5)- to 10(6)-fold less infectious than similar levels of wild-type virus. One mutant, DeltaCys33-Cys36, was also found to be noninfectious in vivo when mutant virus was administered intravenously to a pig-tailed macaque. NC mutations can therefore be used to generate replication defective virions for candidate vaccines in the SIV macaque model for primate lentiviral diseases.

We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins.

Strategies for the treatment of human immunodeficiency virus-type 1 (HIV-1) infection must contend with the obstacle of drug resistance. HIV-1 nucleocapsid protein zinc fingers are prime antiviral targets because they are mutationally intolerant and are required both for acute infection and virion assembly. Nontoxic disulfide-substituted benzamides were identified that attack the zinc fingers, inactivate cell-free virions, inhibit acute and chronic infections, and exhibit broad antiretroviral activity. The compounds were highly synergistic with other antiviral agents, and resistant mutants have not been detected. Zinc finger-reactive compounds may offer an anti-HIV strategy that restricts drug-resistance development.

The zinc fingers of the HIV-1 nucleocapsid protein, NCp7, are prime targets for antiretroviral therapeutics. Here we show that S-acyl-2-mercaptobenzamide thioester (SAMT) chemotypes inhibit HIV by modifying the NCp7 region of Gag in infected cells, thereby blocking Gag processing and reducing infectivity. The thiol produced by SAMT reaction with NCp7 is acetylated by cellular enzymes to regenerate active SAMTs via a recycling mechanism unique among small-molecule inhibitors of HIV.

The presence of relatively high levels of cellular protein contamination in density-purified virion preparations is a confounding factor in biochemical analyses of HIV and SIV produced from hematopoietic cells. A major source of this contamination is from vesicles, either microvesicles or exosomes, that have similar physical properties as virions. Thus, these particles can not be removed by size or density fractionation. Although virions and vesicles have similar cellular protein compositions, CD45 is excluded from HIV-1 yet is present in vesicles produced from hematopoietic cells. By exploiting this finding, we have developed a CD45 immunoaffinity depletion procedure that removes vesicles from HIV-1 preparations. While this approach has been successfully applied to virion preparations from several different cell types, some groups have concluded that "exosomes" from certain T cell lines, specifically Jurkat, do not contain CD45. If this interpretation is correct, then these vesicles could not be removed by CD45 immunoaffinity depletion. Here we show that dense vesicles produced by Jurkat and SupT1/CCR5 cells contain CD45 and are efficiently removed from preparations by CD45-immunoaffinity depletion. Also, contaminating cellular proteins were removed from virion preparations produced by these lines. Previously, the absence of CD45 from both "exosomes" and virions has been used to support the so called Trojan exosome hypothesis, namely that HIV-1 is simply an exosome containing viral material. The presence of CD45 on vesicles, including exosomes, and its absence on virions argues against a specialized budding pathway that is shared by both exosomes and HIV-1.

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6 Gag proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9 NC/SP2 protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8 SP2/p6 or p15 NC/SP2/p6 protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6 Gag site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6 Gag and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9 NC/SP2 species in some aspect of integration.

Human immunodeficiency virus type 1 (HIV-1) infects CD4 + T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both Pr55 Gag expression and full-length proviral constructs. Membrane-permeable biarsenical compounds FlAsH and ReAsH covalently bond to this tetracysteine sequence and specifically fluoresce, effectively labeling Gag in the cell. Biarsenical labeling readily and specifically detected a tetracysteine-tagged HIV-1 Gag protein (Gag-TC) in HeLa, Mel JuSo, and Jurkat T cells by deconvolution fluorescence microscopy. Gag-TC was localized primarily at or near the plasma membrane in all cell types examined. Fluorescent two-color analysis of Gag-TC in HeLa cells revealed that nascent Gag was present mostly at the plasma membrane in distinct regions. Intracellular imaging of a Gag-TC myristylation mutant observed a diffuse signal throughout the cell, consistent with the role of myristylation in Gag localization to the plasma membrane. In contrast, mutation of the L-domain core sequence did not appreciably alter the localization of Gag, suggesting that the PTAP L domain functions at the site of budding rather than as a targeting signal. Taken together, our results show that Gag concentrates in specific plasma membrane areas rapidly after translation and demonstrate the utility of biarsenical labeling for visualizing the dynamic localization of Gag.

Retroviral nucleocapsid (NC) and its Zn2+ fingers are found to be critical for many steps in the viral life cycle. Studies have shown NC's involvement in virion assembly (5, 10, 15, 17-19, 49), reverse transcription (7, 24, 46, 51), and even integration processes (7-9). The Zn2+ finger in NC is one of the most highly conserved elements (4, 11) in the orthoretroviruses (52). NC, as a domain of the Gag precursor, is involved in the assembly of virions and packaging of the genomic RNA. The NC protein, generated during maturation of virions through the action of the viral protease, is involved in the reverse transcription and integration steps of the virus life cycle. The Zn2+ finger structures are critical for the survival of retroviruses, since this and other laboratories have shown that a variety of alterations to the Zn2+ fingers typically result in particles that are replication defective (14, 20, 25, 26, 51). When the Cys or His residues are changed to amino acids that no longer bind Zn2+, virions are defective in RNA packaging and replication. Thus, the highly conserved NC Zn2+ finger structures are critical for assembly and infection processes. It was observed that a number of NC mutant murine leukemia and human immunodeficiency virus type 1 (HIV-1) viruses were still able to package their RNA genomes, and yet they were replication defective (20, 22, 25). NC in these mutants is still able to coordinate significant levels of Zn2+ since the metal-coordinating residues are substituted with other Zn2+ coordinating amino acids (36). The replication defects in these mutants manifest themselves at the level of reverse transcription, since there is a reduction in the level of viral DNA (vDNA) produced upon infection. There is also a major defect in the ability of the these NC mutant virions to protect their vDNA once it is generated (7, 24, 51). In one particular HIV-1 NC mutant (NCH23C), there is also a defect in the ability of virions to mediate the end processing of the vDNA prior to integration, suggesting that NC and its Zn2+ fingers function in the preintegration with the viral integrase protein (7). Thus, one can observe the central importance of NC and the conserved Zn2+ finger structures at various critical stages of the viral life cycle. Surprisingly, there is a class of proteins called cellular nucleic acid binding proteins (CNBPs) that contain seven Zn2+ finger sequences that are very similar to those found in retroviral NC (3, 6, 12, 35, 45, 47, 55). Retroviral NC and CNBP Zn2+ fingers have arrangements of amino acids with the general sequence: “-Cys-φ-X-Cys-Gly-±-X-Gly-His-X3-δ-Cys-,” where X is a variable amino acid, φ is an aromatic residue, “±” is a charged amino acid, and δ is a carbonyl-containing residue. There is absolute amino acid homology between retroviral NC and CNBP Zn2+ fingers among the Cys and His metal ion-binding residues (CCHC) and the two Gly residues listed above. Additionally, there is conservation of the positions of functionally homologous residues within the Zn2+ finger loops with an aromatic residue (φ) in the first loop, a charged residue (±) in the second loop, and a carbonyl-containing amino acid (δ) in the third loop (Table ​(Table1).1). CNBPs from a number of vertebrates are highly homologous at the protein and nucleic acid levels. They have been found in humans (45), rodents (6, 35, 55), chickens (47), and amphibians (3, 12). TABLE 1. Comparison of the HIV-1 NC Zn2+ fingers with the seven Zn2+ fingers from human CNBP The true functions of CNBPs among the various species listed are still under study. Initial reports on mammalian CNBP indicate that it binds to the sterol response element (SRE) (42, 45, 54), which regulates cholesterol levels. Later studies suggested that CNBP was probably not involved in sterol regulation (41, 55, 56). In rodents, CNBP was found to enhance promoter activity of the colony-stimulating factor 1 promoter upon binding (35). A cDNA coding for CNBP has been generated from the mRNA isolated from a wide array of tissues in chickens (47). CNBP was shown to be a translational regulatory factor for ribosomal proteins and is differentially expressed at various stages of development in amphibians (3, 12, 43). Another CNBP was found to interact with JC virus promoter-enhancers (39). There has recently been a report of another human protein that has Zn2+ fingers with most of the characteristics of the retroviral NC Zn2+ finger, which is related to the Mpe1p protein found in yeast (53). As the CNBP and retroviral NC Zn2+ fingers generally appear to function in nucleic acid interactions in their respective systems and have quite similar sequences, we wished to determine whether CNBP Zn2+ fingers could serve as suitable substitutes for the proximal NC Zn2+ finger in HIV-1 NC. In the present study, the seven Zn2+ fingers from human CNBP were individually exchanged into HIV-1 NC, and mutant viruses were characterized. An analysis was also performed to model the Zn2+ finger structures from CNBP and HIV-1 NC in an attempt to identify possible divergent structural features, which may partially explain why one of the mutants was replication defective.

The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10 −6 relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined. We found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane assembly, PPPY L domain) and Mason-Pfizer monkey virus (cytoplasmic assembly, PPPY L domain), similar to the reduction observed for HIV-1. Thus, proteasome inhibitors can affect the budding of a virus that assembles within the cytoplasm. However, the budding of mouse mammary tumor virus (MMTV; cytoplasmic assembly, unknown L domain) was unaffected by proteasome inhibitors, similar to the proteasome-independent budding previously observed for equine infectious anemia virus (plasma membrane assembly, YPDL L domain). Examination of MMTV particles detected Gag-ubiquitin conjugates, demonstrating that an interaction with the ubiquitination system occurs during assembly, as previously found for other retroviruses. For all of the cell lines tested, the inhibitor treatment effectively inactivated proteasomes, as measured by the accumulation of polyubiquitinated proteins. The ubiquitination system was also inhibited, as evidenced by the loss of monoubiquitinated histones from treated cells. These results and those from other viruses show that proteasome inhibitors reduce the budding of viruses that utilize either a PPPY- or PTAP-based L domain and that this effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion.

Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4 + T cells long before a quantitative decline in circulating CD4 + T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4 + T cells remains unclear. Both direct effects of cytopathic infection of CD4 + cells and indirect effects in which uninfected “bystander” cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.

HLA class II DR is one of the most abundant cell surface proteins incorporated onto human immunodeficiency virus type 1 (HIV-1) during budding. The mechanism for HLA class II protein incorporation is not known and may involve a viral protein. To determine whether Env affects HLA class II protein incorporation, HIV-1 virions, either with or without Env on their surface, were produced from HLA class II-expressing cells and analyzed by whole-virus immunoprecipitation with antisera against HLA class II proteins. HLA class II proteins were detected on virions only when wild-type Env was incorporated, while similar experiments showed that HLA class I proteins were incorporated independent of Env packaging. Therefore, the packaging of HIV-1 Env protein is required for the efficient incorporation of HLA class II but not class I proteins into the virion. Analysis of two Env mutants revealed that the presence of a 43-amino-acid sequence between amino acids 708 and 750 in the gp41TMcytoplasmic tail was required for efficient incorporation of HLA class II proteins. These data show that HIV-1 actively incorporates HLA class II proteins in a process that, either directly or indirectly, requires Env.

The presence of FK506-binding protein-12 was demonstrated in virions of HIV-1, although its concentration was lower than that of cyclophilin A. The effect of two inhibitors of the peptidyl-prolyl cis-trans isomerases FK506 and cyclosporin A (CsA) was studied in H9 cells that were chronically infected by HIV-1. Both drugs inhibited virus production in the infected cells in a concentration-dependent manner, by decreasing the number of the producing cells. FK506 did not have an effect on Gag processing, based on the p24 antigen content of virions produced in the presence of this drug. Furthermore, FK506 treatment of uninfected H9 cells did not diminish their susceptibility toward HIV-1 infection, whereas CsA treatment decreased the degree of HIV-1 infection with an IC(50) of 1-2 microg/ml. Also, pretreatment of the virus with CsA decreased its infectivity in HeLaCD4-LTR/beta-gal cells; in contrast, at concentrations up to 10 microg/ml, FK506 did not have an effect. Our findings on the antiviral activity of FK506 and CsA suggest that FK506 is effective only in chronically infected cells, by selectively inhibiting the growth of HIV-1 infected cells, whereas CsA has a specific effect on virus replication.

The p6 Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6 Gag between Y 36 and P 37 , apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6 Gag proteins in HIV-1 MN . To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6 Gag , site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6 Gag , Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41 TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6 Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6 Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12 Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6 Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.

Publisher Summary Recently, it has been shown that Cys (X)2 Cys (X)4 His (X)4 Cys array (CCHC) zinc finger peptides are susceptible to chemical attack by a wide variety of oxidizing agents. The metal-chelated sulfur thiolates in the CCHC zinc fingers of HIV-1 p7 are known to react with a variety of chemical groups, including maleimides, nitrosos, disulfoxides, thiocarbamoyl-disulfides, and other substituted disulfides as well as oxidizing agents, such as Cu +2 , Fe +3 , and Hg +2 ions. The reaction mechanism for the thiuram disulfide class of oxidizing agents and maleimide class of alkylating agents are examined and presented in this chapter. Thiuram disulfides are examined in detail, as a member of this class of compounds, tetraethylthiuram disulfide (Antabuse) is an FDA-approved drug for alcohol abuse therapy and has very low in vivo toxicity. These compounds have functional groups that can modify zinc fingers in nucleocapsid (NC) protein and have antiviral activity but are not necessarily specific for the virus. To initiate studies leading to the design of reagents with greater specificity for the viral NC protein, it is necessary to determine the mechanism of action for model compounds and in particular to determine the initial site of attack on the NC protein. While the studies of NC protein alone demonstrated little if any cross-linking, the results with HIV-1 virus showed extensive oligomerization. The mature virion contains a compact ribonucleoprotein complex formed by the genomic RNA and ca. 2,500 copies of the NC protein. Therefore, the high concentration of NC in the viral particle the formation of intermolecular disulfide bonds over intramolecular ones is expected to be favored following virus treatment with thiuram disulfides.

Previous reports have shown that cyclophilin A (CyPA) is found to be specifically associated with human immunodeficiency virus type-1 (HIV-1) virions and is required for infectivity (Franke et al. Nature 372:359; Thali et al. Nature 372:363). We have examined CyPA associated with HIV-1MN virions. Virions from infected human lymphoid cells were analyzed by high-pressure liquid chromatography (HPLC), protein sequence, and immunoblot analysis. At least three forms of CyPA were found: an unmodified form, an N-terminally modified form, and an N-terminally modified form that migrates as a larger isoform on a reducing-SDS polyacrylamide gel. Using a protease digestion procedure, CyPA that is associated with virions was found to be located inside the viral membrane. Similar examination of SIVMne produced by HUT-78 human T cells did not detect specific incorporation of CyPA into SIV virions. Our results are consistent with the role of CyPA acting early in the infectious process of HIV-1.

CD8(+) cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A()01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B()17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4(+)T cells infected with an engineered SIV(mac)239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4(+)T cells, the TW9 mutant virus failed to induce IFN-gamma expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.

Background The TRIM5α protein is a principal restriction factor that contributes to an HIV-1 replication block in rhesus macaque CD4+ T cells by preventing reverse transcription. HIV-1 restriction is induced in human CD4+ T cells by expression of rhesus TRIM5α as well as those of other old world monkeys. While TRIM5α restriction has been extensively studied in single-round infection assays, fewer studies have examined restriction after extended viral replication. Results To examine TRIM5α restriction of replication, we studied the ability of TRIM5α proteins from African green monkey (AgmTRIM5α) and gorilla (gorTRIM5α) to restrict HIV-1 and SIVmac239 replication. These xenogeneic TRIM5α genes were transduced into human Jurkat-CCR5 cells (JR5), which were then exposed to HIV-1 or SIVmac239. In our single-round infection assays, AgmTRIM5α showed a relatively modest 4- to 10-fold restriction of HIV-1 and SIVmac239, while gorTRIM5α produced a 2- and 3-fold restriction of HIV-1 and SIVmac239, respectively, consistent with the majority of previously published single-round studies. To assess the impact of these modest effects on infection, we tested restriction in replication systems initiated with either cell-free or cell-to-cell challenges. AgmTRIM5α powerfully restricted both HIV-1 and SIVmac239 replication 14 days after cell-free infection, with a ≥ 3-log effect. Moreover, expression of AgmTRIM5α restricted HIV-1 and SIVmac239 replication by 2-logs when co-cultured with infected JR5 cells for 12 days. In contrast, neither expression of gorTRIM5α nor rhesus TRIM5α induced significant resistance when co-cultured with infected cells. Follow up experiments showed that the observed differences between replication and infection were not due to assembly defects as xenogeneic TRIM5α expression had no effect on either virion production or specific infectivity. Conclusions Our results indicate that AgmTRIM5α has a much greater effect on extended replication than on any single infection event, suggesting that AgmTRIM5α restriction acts cumulatively, building up over many rounds of replication. Furthermore, AgmTRIM5α was able to potently restrict both HIV-1 and SIV replication in a cell-to-cell infection challenge. Thus, AgmTRIM5α is unique among the TRIM5α species tested to date, being able to restrict even at the high multiplicities of infection presented by mixed culture with nonrestrictive infected cells.