**2. Brief overview on HIV replication cycle**

The replication cycle of HIV can be divided into five major steps: (i) virus-receptor interactions and fusion; (ii) reverse transcription and proviral integration; (iii) HIV genomic DNA tran‐ scription; (iv) HIV mRNA splicing, nuclear export and translation; and (v) viral assembly, release and maturation (Figure 1).

The first step of the cycle begins with the binding of the virion gp120 surface subunit (SU glycoprotein) to CD4 receptor present in T-cells, macrophages and dendritic cells. The SU glycoprotein and the gp41 transmembrane subunit (TM glycoprotein) remain associated by non-covalent binding. Both SU and TM are proteolytically cleaved from the envelope (Env) precursor protein by a cellular convertase, furin, within the endoplasmatic reticulum (ER). The SU glycoprotein allows viral binding to cellular receptors – CD4 and a coreceptor belonging to the chemokine receptor's family – while the TM protein is involved in the fusion between the viral envelope and the host cell membrane [1]. After initial binding to CD4, SU undergoes structural changes that lead to the exposure (or formation) of the coreceptor-binding site. Although several chemokine receptors were identified as mediators of HIV entry *in vitro*, CCR5 and CXCR4 seem to be the two major coreceptors [2, 3]. After SU glycoprotein binding to coreceptor additional conformational changes are observed, exposing the N-terminal region of TM (dubbed the "fusion peptide"), which mediates the fusion between the viral and host membranes (reviewed in [4, 5]). This viral fusion process may occur through a direct pHindependent fusion mechanism with plasma membrane [6], or via endocytosis and fusion with endosomes [7].

After viral fusion, the viral capsid enters the cytoplasm and the viral RNA is converted to double-stranded DNA, a reaction mediated by the viral reverse transcriptase (RT), that occurs in a cytoplasmic complex named the reverse transcriptase complex (RTC). RT has three essential activities for virus replication: RNA-dependent DNA polymerase (i.e. reverse transcriptase), RNase H activity that cleaves the genomic RNA in RNA/ DNA hybrids during cDNA synthesis, and DNA-dependent DNA polymerase activity (for synthesis of the second strand of the proviral DNA). The result is a double-stranded DNA replica of the original genomic RNA. The double-stranded viral DNA, as part of the preintegration complex (PIC), penetrates the host cell nucleus through the pores in the nuclear membrane. Another viral enzyme, integrase, inserts the double-stranded viral DNA in the host cell chromosomal DNA (reviewed in [8]). The PIC is composed of several cellular and viral components, e.g. viral DNA, RT, integrase (IN), capsid (CA), matrix (MA) and Vpr proteins. In activated cells, the proviral DNA is transcribed, acting as a template for mRNA synthesis. The viral mRNA exists as three distinct classes: multiply spliced (~2kb), single-spliced (4-5kb) and unspliced (9kb). The multiply spliced transcripts are the first to accumulate soon after infection and encode the regulatory proteins Tat, Rev and Nef. The accumulation of Rev protein enables the efficient nuclear export of single-spliced and unspliced mRNA and to an increase in the levels of these mRNAs (reviewed in [9]).

**2. Brief overview on HIV replication cycle**

76 Trends in Basic and Therapeutic Options in HIV Infection - Towards a Functional Cure

release and maturation (Figure 1).

endosomes [7].

mRNAs (reviewed in [9]).

The replication cycle of HIV can be divided into five major steps: (i) virus-receptor interactions and fusion; (ii) reverse transcription and proviral integration; (iii) HIV genomic DNA tran‐ scription; (iv) HIV mRNA splicing, nuclear export and translation; and (v) viral assembly,

The first step of the cycle begins with the binding of the virion gp120 surface subunit (SU glycoprotein) to CD4 receptor present in T-cells, macrophages and dendritic cells. The SU glycoprotein and the gp41 transmembrane subunit (TM glycoprotein) remain associated by non-covalent binding. Both SU and TM are proteolytically cleaved from the envelope (Env) precursor protein by a cellular convertase, furin, within the endoplasmatic reticulum (ER). The SU glycoprotein allows viral binding to cellular receptors – CD4 and a coreceptor belonging to the chemokine receptor's family – while the TM protein is involved in the fusion between the viral envelope and the host cell membrane [1]. After initial binding to CD4, SU undergoes structural changes that lead to the exposure (or formation) of the coreceptor-binding site. Although several chemokine receptors were identified as mediators of HIV entry *in vitro*, CCR5 and CXCR4 seem to be the two major coreceptors [2, 3]. After SU glycoprotein binding to coreceptor additional conformational changes are observed, exposing the N-terminal region of TM (dubbed the "fusion peptide"), which mediates the fusion between the viral and host membranes (reviewed in [4, 5]). This viral fusion process may occur through a direct pHindependent fusion mechanism with plasma membrane [6], or via endocytosis and fusion with

After viral fusion, the viral capsid enters the cytoplasm and the viral RNA is converted to double-stranded DNA, a reaction mediated by the viral reverse transcriptase (RT), that occurs in a cytoplasmic complex named the reverse transcriptase complex (RTC). RT has three essential activities for virus replication: RNA-dependent DNA polymerase (i.e. reverse transcriptase), RNase H activity that cleaves the genomic RNA in RNA/ DNA hybrids during cDNA synthesis, and DNA-dependent DNA polymerase activity (for synthesis of the second strand of the proviral DNA). The result is a double-stranded DNA replica of the original genomic RNA. The double-stranded viral DNA, as part of the preintegration complex (PIC), penetrates the host cell nucleus through the pores in the nuclear membrane. Another viral enzyme, integrase, inserts the double-stranded viral DNA in the host cell chromosomal DNA (reviewed in [8]). The PIC is composed of several cellular and viral components, e.g. viral DNA, RT, integrase (IN), capsid (CA), matrix (MA) and Vpr proteins. In activated cells, the proviral DNA is transcribed, acting as a template for mRNA synthesis. The viral mRNA exists as three distinct classes: multiply spliced (~2kb), single-spliced (4-5kb) and unspliced (9kb). The multiply spliced transcripts are the first to accumulate soon after infection and encode the regulatory proteins Tat, Rev and Nef. The accumulation of Rev protein enables the efficient nuclear export of single-spliced and unspliced mRNA and to an increase in the levels of these

**Figure 1. Schematic representation of HIV replication cycle**. HIV initiates infection by attaching (1) to cellular recep‐ tors: CD4 and a chemokine receptor (co-receptor). The interactions with both receptors trigger the fusion between viral envelope with cellular membrane, either after endocytosis (2A) or by direct fusion with plasma membrane (2B). The release of viral nucleocapside into the cytoplasm (3) precedes the formation of the reverse transcriptase complex (RTC)

where the reverse transcription takes place (4). The RTC transforms to the preintegration complex (PIC), composed by several cellular and viral components, that is imported to the nucleus where viral DNA is integrated into cellular chro‐ mosomal DNA (5 and 6). The proviral DNA is then transcribed (7) and mRNA migrates to the cytoplasm and translat‐ ed to viral proteins (8). Assembly of different components of viral particles occurs at plasma membrane (9). After egress and release of immature virions (10), the proteolytical cleavage of Gag polyprotein takes place leading to ma‐ ture virions (11).

After replication, transcription and translation, the viral genome information is ready to proceed to the final step: the viral assembly, the release and maturation of recently formed virions. The nucleocapsid assembly occurs through protein-protein interactions mediated by the uncleaved Gag polyprotein – through the capsid (CA) domain [10] – that also recruits the viral genomic RNA, through the interaction between the nucleocapsid (NC) domain and the RNA packaging signal (*Psi* sequence) [11]. The NC domain also mediates the formation of the RNA dimer via a palindromic sequence in the dimer linkage structure (DLS) sequence, which is located in the *Psi* sequence. In addition, specific cellular tRNAs are packaged. The assembly of the virus particle, which final steps occur at the plasma membrane (reviewed in [12]), is partly regulated by the Vpu and Vif proteins, which play an important role in the assembly of the virus. At the cell membrane, the immature viruses are released and maturation takes place through polypeptide cleavage mediated by the viral protease. The mature virus is now able to infect other cells.
